Generation of urine-derived induced pluripotent stem cells from a patient with phenylketonuria

Abstract

The aim of the study was to establish an induced pluripotent stem cell line from urine-derived cells (UiPSCs) from a patient with phenylketonuria (PKU) in order to provide a useful research tool with which to examine the pathology of this rare genetic metabolic disease. Urine-derived epithelial cells (UCs) from a 15-year-old male patient with PKU were isolated and reprogrammed with integration-free episomal vectors carrying an OCT4, SOX2, KLF4, and miR-302-367 cluster. PKU-UiPSCs were verified as correct using alkaline phosphatase staining. Pluripotency markers were detected with real-time PCR and flow cytometry. Promoter methylation in two pluripotent genes, and, was analyzed using bisulphite sequencing. An embryoid body (EB) formation assay was also performed. An induced pluripotent stem cell line (iPSC) was generated from epithelial cells in urine from a patient with PKU. This cell line had increased expression of stem cell biomarkers, it efficiently formed EBs, it stained positive for alkaline phosphatase (ALP), and it had a marked decrease in promoter methylation in the and genes. The PKU-UiPSCs created here had typical characteristics and are suitable for further differentiation.

Urine-derived induced pluripotent/neural stem cells for modeling neurological diseases

“…Another widely used method for iPSC reprogramming is Sendai virus, which is a single stranded negative sense RNA virus that does not integrate into the genome. Sendai reprogramming has been used to generate UiPSCs from the urine of patients with attention deficit hyperactivity disorder (ADHD) [ 67 ], obsessive–compulsive disorder [ 68 ], Duchenne muscular dystrophy [ 69 – 71 ], dilated cardiomyopathy [ 72 ], heterozygous PAI-I mutation [ 73 ], ventricular septal defect (VSD) [ 74 ], X-linked Alport syndrome (X-LAS) [ 75 ], spinal cord injury [ 76 ], type 2 diabetes mellitus [ 77 ], and healthy donors [ 78 ]. The majority of the iPSC colonies were undetectable for Sendai virus transgenes after 7 passages [ 67 ].…”

“…Another study investigated PCSK9-mediated autosomal dominant hypercholesterolemia (ADH) using UiPSCs generated by episomal vectors coding for OCT4, SOX2, KLF4, MYC, LIN28, NANOG and SV40LT, and a non-episomal vector coding for miR302/367 [ 43 ]. In addition, integration-free UiPSC lines from individuals with Down syndrome (DS) [ 84 ], Phenylketonuria (PKU) [ 73 ] and Type 2 long QT syndrome [ 85 ] have been successfully established via episomal technology.…”

Urinary Stem Cells as Tools to Study Genetic Disease: Overview of the Literature

“…This model could overcome the issues associated to both the lack of an appropriate disease model and the difficulty in obtaining neural cells. Moreover, the differentiation of iPSCs into osteoblasts and osteoclasts could allow the study of mechanisms underlying bone impairment in PKU in order to identify new targets and treatments [39].…”

Urine-derived stem cells: Promising advancements and applications in regenerative medicine and beyond

“…Finally, confirmation of chondrogenic, osteogenic and adipogenic differentiation further constitutes an integral aspect of the research endeavor. While initial investigations predominantly centered on USC extraction from healthy populations, ongoing research expanded the scope to encompass diverse pathological conditions, including acute kidney injury, hemophilia A (HA), phenylketonuria (PKU), Down syndrome (DS), and X-linked Alport syndrome (X-LAS) [ [15] , [16] , [17] , [18] , [19] ]. Notably, individual USCs exhibit impressive expansion capabilities, with the capacity to multiply up to 60–70 times while maintaining long telomeres.…”